<p>Immunoglobulin Y (IgY) is the major antibody found in eggs from chicken (Gallus domesticus). IgY can be used as an alternative to mammalian antibodies normally used in research, and its use in immunotherapy has recently been proposed. Compared to mammalian antibodies, IgY possesses several biochemical advantages and its simple purification from egg yolk prevents a stressful moment in animal handling, as no bleeding is necessary. </p><p>Small amount of antigen (1 mg) can be used to elicit an immune response in chickens and there are low intra-individual differences regarding antibody concentration found in yolk. By studying two chicken breeds and their cross, a genetic correlation was shown regarding the IgY concentration, which implies a possibility by breeding to increase IgY concentrations. By using IgY instead of goat antibody as capture antibody in ELISA, it is possible reduce interferences by complement activation. After oral administration of IgY to healthy volunteers, IgY activity was present in saliva 8 hours later, indicating a protective effect. This effect has been studied in an open clinical trial with cystic fibrosis patients. Specific IgY against Pseudomonas aeruginosa given orally prolongs the time of intermittent colonization by six months, decrease the number of positive colonizations and might be a useful complement to antibiotic treatment. Immunoglobulin therapy may diminish the development of antibiotic resistant microorganisms. The use of immunoglobulin therapy broadens the arsenal available to combat pathogens in medicine and IgY is a promising candidate, both as an alternative to antibiotics and as a useful tool in research and diagnostics.</p>

<p>Microarray technology is a promising approach for the simultaneous analysis of multiple single nucleotide polymorphisms (SNPs), which are the most abundant form of genetic variation. In this thesis enzyme-assisted microarray-based methods were developed to improve the accuracy and genotype discrimination power of the current methods for SNP genotyping. The improved technology was applied for analysing recessively inherited disease mutations, for Y-chromosomal SNPs in a population study, for an evolutionary analysis of SNPs in flycatchers and for multiplexed quantitative determination of SNP-allele frequencies in pooled DNA samples. </p><p>A robust attachment chemistry for immobilising oligonucleotides on glass surface was established, based on an evaluation of eight covalent coupling methods. A four-colour fluorescence detection strategy, which enabled a multiplexed quantitative analysis for as little as 2% of a minority allele frequency in pooled samples was generated. </p><p>Twenty-five Y-chromosomal SNPs were screened in a collection of 300 samples from five Finno-Ugric-speaking populations using minisequencing on microarrays. In these populations six distinct haplotypes were defined by the six SNPs that were polymorphic. Data from five microsatellite markers was combined with the SNP data, revealing shared Y-chromosomal haplotypes between the Finns and the Saami, indicating, in accordance with earlier data, at least two founding Y-chromosomal lineages in these populations.</p><p>Database screening and subsequent validation of 125 potential SNPs in the highly repetitive type 1 interferon genes and genes coding for proteins in the interferon-related regulatory pathways revealed 25 informative SNPs in the Finnish and Swedish populations. These SNPs were included in a panel for microarray based genotyping that should find a variety of applications in genetic studies due to the important immunoregulatory functions of the IFN family.</p><p>The significance of sex-chromosome evolution on speciation was investigated in two naturally hybridising flycatcher species (N=459) by analysing a panel of 20 SNPs using minisequencing on microarrays. A strong selection against gene flow across the species boundary of sex-linked genes was observed, as well as a sex-chromosomal influence on male plumage characteristics that have previously been shown to reinforce isolation in these birds. The results suggest a major role for sex-chromosome-mediated isolation of the two flycatcher species.</p>

<p>Diabetes mellitus (DM) is one of the leading causes of end stage renal failure. An increased susceptibility to renal ischemia/reperfusion (I/R)-injury was found in DM rats. Unilateral renal ischemia for as short as 20 minutes led to an irreversible progressive injury in DM kidneys, whereas the injury in non-DM kidneys was almost reversible. The renal I/R injury was characterized by anuria, infiltration of inflammatory cells, tubular atrophy, dilation of the remaining tubuli and tubulointerstitial fibrosis. Necrotic areas were found in the inner parts of the outer medulla and in the papilla. The renal medulla was the most vulnerable part of the kidney. This was seen both by the extent of fibrosis four and eight weeks after I/R and by the presence of TUNEL-positive (apoptotic) cells 6h after ischemia. Increased accumulation of HA and enhanced CD44 expression was seen after I/R in DM kidneys.</p><p>Treatment with long acting insulin 7-14 days before I/R, decreased the number of apoptotic cells in the renal medulla and protected renal function and morphology after the insult, while insulin treatment after the injury did not have any protective effect. Short acting insulin given 2-6 hours before I/R partially protected renal function but did not improve the morphological picture.</p><p>Treatment with the angiotensin II receptor type 1 blocker candesartan, the PAF-antagonist UR-12670, the immunosuppressive agents tacrolimus and cyclosporin A, or prednisolone did not improve the outcome of the renal I/R injury in DM. Injection of cobalt protoporphyrin (CoPP) intraperitoneally in order to induce an over-expression of heme oxygenase-1 (HO-1) resulted in a trend towards a better function in DM kidneys after I/R. However, the induction of HO-1 by intraperitoneal CoPP injection was not achieved in all rats, when examined by western blot.</p><p>In conclusion, unilateral renal I/R leads to a severe progressive injury in DM kidneys. Insulin treatment before ischemia, but not after, reduces the renal injury in DM rats. Studies using a more reliable administration of CoPP are required to decide if induction of HO-1 protects against renal I/R injury in DM.</p>

<p>The eosinophil cationic protein (ECP) is usually associated with the eosinophil granulocyte. In this thesis the presence and production of this protein has been studied in two other cells. The circulating monocyte was found to contain ECP mRNA and small amounts of ECP, one thousand times less than that found in the eosinophil. The production decreased by differentiation of the myelomonoblastic cell line U937 into a macrophage phenotype. Submucosal lung macrophages did not stain for ECP and alveolar macrophages did not contain ECP mRNA. The circulating neutrophil contains ECP at a level hundred fold less than the eosinophil. We found that the protein is located to the primary granules of the neutrophil but could detect no ECP mRNA in the cell. It was shown in vitro that the protein was taken up by the cell and partly transported to the primary granules. The uptake did not seem to be receptor mediated. Upon stimulation of the neutrophils, ECP previously taken up, was re-secreted. </p><p>The ECP protein is heterogeneous both to molecular characteristics and to function. To evaluate if a genetic component is involved, the ECP gene was analysed in 70 individuals. Three single nucleotide polymorphisms (SNP´s) were found, denoted 277(C>T), 434(G>C) and 562(G>C). The two first were located to the mature peptide-coding region and would change the amino acids, arg45cys and arg97thr. The prevalence of the most common SNP, 434, was evaluated in two eosinophil-related diseases, allergy/asthma and Hodgkin Lymphoma (HL). Forty-three HL patients were evaluated and it was found that the 434GG was significantly more prevalent in patients having nodular sclerosis (NS) as compared to other histologies (p=0.03). Erythrocyte sedimentation rate was also related to the 434GG genotype (p=0.009). In 209 medical students 434GG was more common (p=0.002) in those who indicated allergy. The genotype was unrelated to the production of IgE antibodies to allergens. In analysis of 76 subjects with asthma it was found that the 434GG genotype was significantly more common among allergic asthmatics (p=0.04). Asthma and HL-NS are characterized by fibrosis and eosinophils and ECP has been suggested in fibrosis development. </p>

<p>In unstable coronary artery disease (CAD) it still is a matter of debate which patients should undergo early revascularisation. In the FRISC II study (n=2457) an early invasive strategy was, compared to a primarily non-invasive strategy, associated with reduced mortality and myocardial infarction (MI) rates. However, in this heterogeneous group of patients, tools for an appropriate selection to revascularisation are needed.</p><p>From the FRISC II study we evaluated the prognosis, the angiographic extent of CAD and the effects of an early invasive strategy in relation to risk variables on admission.</p><p>The occurrence of ST depression and/or elevated levels of Troponin T were associated with a higher risk for death and MI, more severe CAD and also with a reduction of death or MI by the early invasive strategy.</p><p>Elevated levels of the inflammatory markers C-reactive protein (CRP) and interleukin-6 (Il-6) were associated with a higher mortality but an unchanged MI rate. Elevated levels of Il-6, but not CRP, identified patients with a large reduction of mortality by the invasive strategy.</p><p>Age ≥ 70 years, male gender, diabetes, previous MI, ST depression and elevated levels of troponin and markers of inflammation were independently associated with an adverse outcome. The FRISC-score was constructed using these 7 variables. At FRISC-score ≥ 5 an early invasive strategy markedly reduced mortality and MI, at FRISC–score 3-4 death/MI was reduced, whereas in patients with a FRISC-score 0-2 neither mortality nor death/MI was influenced.</p><p>In unstable CAD, a non-invasive strategy seems justified only for patients at low risk, i.e. FRISC score < 2. In patients with intermediate and high risk, i.e. FRISC-score ≥ 3, an early invasive strategy is recommended.</p>

<p>Parathyroid hormone (PTH) is the major physiological regulator of the extracellular Ca²⁺ concentration ([Ca²⁺]_o) in the body. The secretion of this hormone is suppressed at high [Ca²⁺]_o. Previously this was thought to occur by intracellular degradation of the hormone in the secretory pathway of parathyroid (PT) cells but is now believed to result from extracellular Ca²⁺ stimulus-secretion coupling via the calcium sensing receptor (CaR). In contrast to the stimulation of PTH secretion upon inhibition of mature PTH proteolysis, inhibition of PT proteasomes caused the accumulation of PTH precursors and inhibited secretion of PTH. This suggests that PT proteasomes play a quality control function in the maturation of PTH but they do not directly participate in the [Ca²⁺]_o-regulated secretion of the hormone. Treatment of PT cells with 12-O-tetradecanyolphorbol-13-acetate (TPA) blocks the high [Ca²⁺]_o-induced CaR-mediated suppression of PTH secretion. To delineate the role of DAG-responsive protein kinase C (PKC) isoforms in this process, we complemented pharmacological modulation of PKC activity with physiological activation of the enzyme via the CaR. PKC-α was rapidly activated by high [Ca²⁺]_o and was efficiently down-regulated by prolonged TPA treatment. In CaR-transfected HEK293 cells, TPA and high [Ca²⁺]_o induced the activation of ERK1/2 but the TPA effect was CaR- and Ca²⁺-independent. The magnitude of neomycin-induced release of Ca²⁺ from intracellular stores following pharmacological modulation of PKC activity was opposite to that resulting from physiological activation/inhibition of the enzyme via the CaR. Influx of Ca²⁺ following activation of the receptor occurred by store-operated mechanisms. Over-expression of wt or DN PKC-α or-ε in PT cells using the Tet-On adenovirus gene delivery system revealed that the stimulatory effect of TPA on PTH secretion at high [Ca²⁺]_o was enhanced in cells over-expressing wt PKC-α, but the coupling of the extracellular Ca²⁺ signal to PTH secretion was not dependent on the physiological activation of this PKC isoform via the CaR.</p>

<p>Serum levels of eosinophil granule proteins namely ECP, EPO and EPX, which are stored in the matrix of specific granules, were shown to correlate with the course of disease in disorders involving eosinophils. The concentration of eosinophil proteins in serum is the result of their release <i>in vivo</i> and <i>ex vivo</i> during the sampling procedure. Generally, eosinophils release the content of their specific granules in three ways: exocytosis, piecemeal degranulation (PM) or cytolysis. Which of them is operating in circulating eosinophils has not yet been defined. The aim of this thesis was to study the mechanisms of granule protein release from blood eosinophils in respect of protein subcellular localization and cell ultrastructure.</p><p>In patients with bacterial infections, serum levels of ECP but not EPO increased, while in patients with viral infections both proteins remained within the range of healthy controls. G-CSF is a cytokine involved in the response mechanism to bacterial but not viral infections. Administration of G-CSF to healthy subjects induced an elevation of eosinophil numbers and a preferential increase of serum EPX and ECP in comparison to EPO.</p><p>The model of PM consists of the stepwise transportation of specific granule contents from the granules towards the plasma membrane. We observed that administration of G-CSF to healthy subjects and the allergen exposure of allergic subjects during the pollen season, caused changes in the ultrastructure of eosinophil specific granules such as loosening of the matrix, granule matrix lucency and ragged losses of their core. Similar alterations of morphology had been previously described for eosinophils undergoing PM.</p><p>ECP, EPX and EPO were localized not only in the specific granules but also in extra-granular compartments as shown both by immuno electron microscopy and subcelular fractionations, An extra-granular EPX compartment was present in healthy as well as in allergic and in hypereosinophilic subjects, and there were no significant differences in its size between the groups. The size of the extra-granular compartments of ECP and EPO was increased in allergics during the season, and these compartments were clearly separate from that of EPX. Results of this show the differential mobilization ofgranule proteins in blood stream eosinophils serum and indicates PM as its mechanism.</p>

<p>In this thesis the oxidative metabolism by blood eosinophils from birch pollen allergic subjects was studied and compared to that by eosinophils from healthy controls, during and out of the pollen season. The effects and mechanisms of <i>in vitro</i> IL-5 priming on blood eosinophils were investigated and compared to the effects of <i>in vivo</i> priming during pollen exposure.</p><p>The main findings of this work were that the oxidative metabolism by blood eosinophils taken from pollen allergic subjects is reduced during the pollen season. The eosinophils taken from asymptomatic allergics have a reduced capacity to produce oxygen free radicals as compared to non-allergic controls. The oxidative metabolism by blood eosinophils from allergic subjects is primed <i>in vivo</i> during the pollen season, as compared to the healthy controls and as compared to out of season. IL-5 primed the oxidative metabolism by eosinophils from allergic subjects in a similar way as eosinophils from healthy controls, both during and out of pollen exposure. The total and tyrosine phosphorylation patterns obtained were identical in eosinophils from allergic subjects and non-allergic controls during the pollen season. Spontaneous phosphorylation was the same in both groups and different from that after IL-5 priming. The oxidative metabolism of blood eosinophils is composed of different stages. The initial stage, measured as the t_½rises of the CL curves, is an indication of the state of priming of the cell, while the end stage, measured as the peaks of the CL curves, is an estimate of the total radical production by the cells. IL-5 priming affected these two stages differently and the two stages are regulated by different signal transduction pathways and IL-5 priming causes a by-passing of MEK.</p><p>In conclusion, in this thesis it is shown that blood eosinophils from allergic subjects are primed <i>in vivo</i> during exposure to their allergen. This <i>in vivo</i> priming leads on one hand to a reduced oxidative metabolism during the pollen season, but also to a faster onset of radical production as a response to certain stimuli. Our data do not provide any evidence of IL-5 involvement in the <i>in vivo</i> priming of blood eosinophils from allergic patients during pollen exposure.</p>

<p>This study evaluated the use of multi-lead ST-monitoring in the early assessment of patients with suspected or confirmed unstable coronary artery disease (UCAD).</p><p>At continuous 12-lead ECG (c12ECG), the definition of an ischemic episode as a transient ST-deviation ¡Ý0 for at least 1 minute resulted in a good observer agreement (kappa=0.72) and an acceptable incidence of postural ST-changes.</p><p>When c12ECG was performed from admission and for 12 hours in 630 patients with suspected UCAD, 16% had ischemic episodes. At 30 days, patients with episodes had a higher risk of cardiac death or myocardial infarction (MI) (10% vs. 1.5%). In a multivariate analysis, troponin T¡Ý0.10¦Ìg/l and presence of ischemic episodes were independent predictors of cardiac death or MI. When ST-monitoring and troponin T status were combined, patients could be divided into a low-, intermediate-, and high-risk group with 1%, 4% and 12% risk for cardiac death or MI at 30 days of follow up.</p><p>As a part of a multicenter trial, including patients with UCAD, 1016 patients underwent ST-monitoring with c12ECG or continuous vectorcardiography (cVCG). Ischemia was detected in 32% and 35%, respectively. When the groups with ischemia were compared, the groups were similar with respect to several clinical variables. Thus, these methods identify the same high-risk population.</p><p>Of the 629 patients treated non-invasively with extended treatment of low-molecular- weight heparin (LMWH) or placebo, 34% had ischemic episodes. In this group at 3 months, patients administered LMWH had a significantly lower risk of death, MI, or revascularization than patients treated with placebo (35.2% vs. 53.4%). In patients without transient ischemic episodes, the outcome in the LMWH and placebo group was similar.</p><p>Thus, multi-lead monitoring provides important prognostic information early after admission in this population, and seems to identify patients who benefit most from extended antithrombotic treatment.</p>

<p>The accumulation of eosinophil granulocytes (EOS) at sites of inflammation is a common feature of astma, allergic rhinitis and inflammatory bowel disease. The aim of the present investigation was to study the mechanisms involved in this accumulation.</p><p>Bronchoalveolar lavage (BAL) fluid obtained from patients with birch-pollen allergy lavaged during season exhibited increased eosinophil chemotactic activity compared with pre-season BAL fluid from the same patients. We identified IL-5, IL-8 and RANTES as the main eosinophil chemotactic agents in the BAL fluid. Only EOS from allergic donors responded to IL-8. IL-2 inhibited albumin-stimulated eosinophil migration towards buffer or chemoattractants. EOS from allergic subjects were less sensitive to this inhibition than EOS from normal subjects, and in vitro priming of the EOS with IL-5 prevented the inhibitory effect of IL-2. We therefore hypothesise that IL-2 acts as an autocrine regulator of EOS migration, and that this inhibitory effect may be down-regulated in allergy, resulting in increased migration of EOS towards chemotactic factors. The stimulation of eosinophil migration by albumin is mediated by PI3 kinase. Decreased expression of CD49d and CD49f caused by albumin may decrease the adhesiveness of the EOS, which in turn may facilitate migration. We found a higher chemotactic activity in perfusion fluids from patients with ulcerative colitis than from control patients. The chemotactic activity correlated with the concentrations of eosinophil granule proteins in the perfusion fluids. IL-5 and TNF-α were identified as two of the chemotactic agents in the perfusion fluid that were inhibited by steroid treatment. Agents with steroid-insensitive chemotactic activity remain to be identified.</p>